The broad, long-term goal of this proposal is the identification and understanding of the regulation of virulence determinants of Borrelia burgdorferi, the agent of Lyme disease. We have chosen to explore as potential virulence factors, antigens that satisfy the following criteria. These antigens are 1) induced in response to an up-shift in culture temperature, such as occurs during a tick blood feeding event leading to the infection of the mammalian host, 2) are the targets of an early humoral response in an infected rhesus monkey, and 3) are absent in non-infectious high-passage variants. As many as 13 different proteins from the low-passage Sh-2-82 strain of B. burgdorferi were found to satisfy this definition of a potential virulence factor. As a necessary prelude to circumvent the confounding problem of genetic heterogeneity, especially as it related to understanding the molecular basis for the differential expression of these Temperature-regulated proteins (Trps), clonal populations of Sh-2-82 exhibiting either Trp+, or Trp- phenotypes, were derived from the low, and high passage variants, respectively, Additionally, the expression of the Trps in these Trp+ clones was confirmed to be similarly temperature dependent as the parental uncloned isolate. Using a representative Trp+ clone as a model, the genes encoding the Trps will be cloned and characterized, and